Fig 2: ABL503 induces 4-1BB signaling only in the context of PD-L1 engagement, which also blocks PD-1/PD-L1 signaling. (A) The luciferase reporter NF-kB-Luc2/4-1BB Jurkat cell line was cultured in the presence of the indicated treatments without target cells. Luciferase activity was measured by relative luminescence units (RLU). (B) Mean fluorescence intensity (MFI) shows the basal expression of PD-L1 in the Panc-1, HCC1954, and RKO cell lines. (C) Induction of 4-1BB signaling was observed when Panc-1, NCI-N87, MDA-MB-231, HCC1954, and RKO cells were co-cultured with reporter Jurkat cells and certain antibodies. (D) A PD-1/PD-L1 blockade bioassay (Promega) was performed to estimate the ability of ABL503 to block PD-1/PD-L1 signaling. (E,) A PD-L1/4-1BB combination bioassay (Promega) was performed, confirming the bispecific effect of ABL503. The EC50 in C, D, and E is derived from ABL503 group. (F) Human peripheral blood mononuclear cells and HCC1954 cells expressing PD-L1 were cocultured with the indicated antibodies. IFN-γ production was assessed by ELISA using cultured supernatant. All samples were duplicated or triplicated and the figures show the representative of three or four independent experiments. Significance was estimated by an unpaired one-way ANOVA with Tukey’s multiple comparisons test using the area under the curve (AUC). The EC50 of ABL503 indicates the mean. Data are presented as mean±SEM. N.S., not significant. **p<0.01; ****p<0.0001.

Fig 3: Novel tumor-targeting anti-PD-L1×4-1BB bispecific antibody (ABL503) simultaneously binds unique sites on its targets. (A) ABL503 was designed to target PD-L1 and 4-1BB at the F(ab)2 and Fc portions, respectively, which prevents the Fc portion from binding FcγR. (B) Schematic illustrating the mechanism of action of ABL503. (C) Surface plasmon resonance confirmed the binding of ABL503 to the targets 4-1BB (left) and PD-L1 (right) at the indicated concentrations. (D) Dual-antigen capture ELISA showed that ABL503 simultaneously bound 4-1BB and PD-L1 at the indicated antibody concentrations. The EC50 of ABL503 indicates the mean. (E) The capacity of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) was estimated by ADCC reporter bioassay during ABL503 treatment. The complement-dependent cytotoxicity (CDC) efficacy of ABL503 was also measured by CDC assay. All experiments were performed by duplicated or triplicated samples.

Fig 4: ABL503 treatment in vivo exerts anti-tumor effects, and recovered mice maintains an anti-tumor response after complete tumor regression. (A) Humanized transgenic mice expressing human PD-L1 and 4-1BB (n=7 per group) were subcutaneously inoculated with MC38hPD-L1 tumor cells. At 6 days after tumor cell injection, when the mean tumor size reached approximately 120 mm3, tumor-bearing mice were randomly allocated to different groups and treated with the indicated antibodies four times at a 3-day interval. (B, C) Tumor volume and survival rate were assessed for the mice described in (A). Tumor growth is presented as mean±SEM, and the significance was calculated by mixed-effects model with Tukey’s multiple comparison test (B and C). N.S., not significant. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. The survival rate was analyzed by the log-rank (Mantel-Cox) test for survival curve comparisons with the control group. N.S., not significant. *p<0.05; **p<0.01; ***p<0.001. (D) At 40 days after primary tumor injection, the recovered group described in C (n=9, including two mice from the 2 mg/kg group, and seven mice from the 10 mg/kg group) was rechallenged using the same tumor cells. Tumor size was measured, and the significance of tumor growth was estimated by mixed-effects model. ****p<0.0001.